D‐amino acid oxidase (DAAO)‐catalyzed selective oxidative deamination is a very promising process for synthesizing l‐amino acids including l‐phosphinothricin ( l‐PPT, a high‐efficiency and broad‐spectrum herbicide). However, the wild‐type DAAO's low activity toward unnatural substrates like d‐phosphinothricin ( d‐PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d‐PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high‐throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d‐PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate‐binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding‐model index to predict the enhanced activity of DAAO toward d‐PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l‐PPT with remarkable efficiency.

中文翻译：

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开发生长选择系统和 d-氨基酸氧化酶袖珍工程以增强 d-膦丝菌素的选择性脱氨活性


D-氨基酸氧化酶（DAAO）催化的选择性氧化脱氨是合成 L-氨基酸（包括 L-草胺膦（l-PPT，一种高效广谱除草剂））的非常有前途的过程。然而，野生型 DAAO 对 d-膦丝菌素 (d-PPT) 等非天然底物的低活性阻碍了其应用。在此，筛选并设计了来自秀丽隐杆线虫的 DAAO (CeDAAO)，以提高 d-PPT 的催化潜力。首先，我们设计了一种新颖的生长选择系统，考虑到大肠杆菌（E. coli）的生长与 DAAO 催化机制之间的复杂关系。开发的系统用于基因库的高通量筛选，结果发现了一种对 d-PPT 催化活性显着增加的变体 (M6)。该变体在具有不同疏水性和亲水性的基材上表现出不同的催化特性。使用Alphafold2建模和分子动力学模拟的分析表明，活性增强的原因是底物结合口袋尺寸增大和合适的电荷分布。进一步的QM/MM计算表明，提高活性的关键因素在于降低还原半反应的初始能垒。最后，开发了一个综合的结合模型指数来预测 DAAO 对 d-PPT 的增强活性，并开发了一种酶促去消旋化方法，使得 l-PPT 的高效合成具有显着的效率。